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GBM Derived Gangliosides Induce T Cell Apoptosis through Activation of the Caspase Cascade Involving Both the Extrinsic and the Intrinsic Pathway.

Mahata B, Biswas S, Rayman P, Chahlavi A, Ko J, Bhattacharjee A, Li YT, Li Y, Das T, Sa G, Raychaudhuri B, Vogelbaum MA, Tannenbaum C, Finke JH, Biswas K - PLoS ONE (2015)

Bottom Line: GBM-ganglioside induced activation of the effector caspase-3 along with both initiator caspases (-9 and -8) in T cells while both the caspase-8 and -9 inhibitors were equally effective in blocking apoptosis (60% protection) confirming the role of caspases in the apoptotic process.Ganglioside-induced T cell apoptosis did not involve production of TNF-α since anti-human TNFα antibody was unable to protect T cells from nuclear blebbing and subsequent cell death.Thus, GBM-ganglioside can mediate T cell apoptosis by interacting with the TNF receptor followed by activation of both the extrinsic and the intrinsic pathway of caspases.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Medicine, Bose Institute, Kolkata, India.

ABSTRACT
Previously we demonstrated that human glioblastoma cell lines induce apoptosis in peripheral blood T cells through partial involvement of secreted gangliosides. Here we show that GBM-derived gangliosides induce apoptosis through involvement of the TNF receptor and activation of the caspase cascade. Culturing T lymphocytes with GBM cell line derived gangliosides (10-20 μg/ml) demonstrated increased ROS production as early as 18 hrs as indicated by increased uptake of the dye H2DCFDA while western blotting demonstrated mitochondrial damage as evident by cleavage of Bid to t-Bid and by the release of cytochrome-c into the cytosol. Within 48-72 hrs apoptosis was evident by nuclear blebbing, trypan blue positivity and annexinV/7AAD staining. GBM-ganglioside induced activation of the effector caspase-3 along with both initiator caspases (-9 and -8) in T cells while both the caspase-8 and -9 inhibitors were equally effective in blocking apoptosis (60% protection) confirming the role of caspases in the apoptotic process. Ganglioside-induced T cell apoptosis did not involve production of TNF-α since anti-human TNFα antibody was unable to protect T cells from nuclear blebbing and subsequent cell death. However, confocal microscopy demonstrated co-localization of GM2 ganglioside with the TNF receptor and co-immunoprecipitation experiments showed recruitment of death domains FADD and TRADD with the TNF receptor post ganglioside treatment, suggesting direct interaction of gangliosides with the TNF receptor. Further confirmation of the interaction between GM2 and TNFR1 was obtained from confocal microscopy data with wild type and TNFR1 KO (TALEN mediated) Jurkat cells, which clearly demonstrated co-localization of GM2 and TNFR1 in the wild type cells but not in the TNFR1 KO clones. Thus, GBM-ganglioside can mediate T cell apoptosis by interacting with the TNF receptor followed by activation of both the extrinsic and the intrinsic pathway of caspases.

No MeSH data available.


Related in: MedlinePlus

GBM derived gangliosides induce apoptosis of T cells.Peripheral blood T lymphocytes were isolated and purified from blood of healthy volunteers by negative selection. T lymphocytes were co-cultured with 15μg/ml of CCF52 derived gangliosides for 18–72 hrs, followed by annexinV-PE/7AAD staining for flow cytometric estimation of apoptosis, as shown in Fig 1A (*p<0.05 vs Media, ***p<0.001 vs Media). Purified T cells were incubated with different concentration (1–10μg/ml) of CCF52 gangliosides for 48hrs and 72hrs and apoptosis was measured by flow cytometric analysis using annexinV-PE/7AAD staining as indicated in Fig 1B (*p<0.05 vs Media, ***p<0.001 vs Media). Fig 1C shows graphical representation showing the percentage of nuclear blebbing event in T cells treated with gangliosides (15μg/ml) isolated from different glioblastoma cell lines for 72hrs (*p<0.05 vs Media, ***p<0.001 vs Media). Fig 1D shows representative photomicrograph demonstrating induction of apoptosis in T cells following 72hrs treatment with three different GBM derived gangliosides (15μg/ml) as evidenced by microscopic evaluation of nuclear blebbing by staining with DAPI. Data represents mean of at least 3 independent experiments unless mentioned otherwise.
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pone.0134425.g001: GBM derived gangliosides induce apoptosis of T cells.Peripheral blood T lymphocytes were isolated and purified from blood of healthy volunteers by negative selection. T lymphocytes were co-cultured with 15μg/ml of CCF52 derived gangliosides for 18–72 hrs, followed by annexinV-PE/7AAD staining for flow cytometric estimation of apoptosis, as shown in Fig 1A (*p<0.05 vs Media, ***p<0.001 vs Media). Purified T cells were incubated with different concentration (1–10μg/ml) of CCF52 gangliosides for 48hrs and 72hrs and apoptosis was measured by flow cytometric analysis using annexinV-PE/7AAD staining as indicated in Fig 1B (*p<0.05 vs Media, ***p<0.001 vs Media). Fig 1C shows graphical representation showing the percentage of nuclear blebbing event in T cells treated with gangliosides (15μg/ml) isolated from different glioblastoma cell lines for 72hrs (*p<0.05 vs Media, ***p<0.001 vs Media). Fig 1D shows representative photomicrograph demonstrating induction of apoptosis in T cells following 72hrs treatment with three different GBM derived gangliosides (15μg/ml) as evidenced by microscopic evaluation of nuclear blebbing by staining with DAPI. Data represents mean of at least 3 independent experiments unless mentioned otherwise.

Mentions: T cells were treated with gangliosides (15μg/ml) isolated from the GBM cell line CCF52 at varying time intervals (Fig 1A) or with varying ganglioside concentration (Fig 1B), followed by staining with annexinV and 7-AAD prior to FACS analysis. CCF52 gangliosides induced time dependent T cell apoptosis, as evidenced by increased annexinV+/7-AAD+ staining at 18hrs (20%) and reaching peak at 72hrs (32%) (Fig 1A). However, significant T cell apoptosis was only observed at 10μg/ml or higher concentration as shown in Fig 1B. Although, CCF52 gangliosides did not show any significant induction in T cell apoptosis at lower concentrations, both GBM gangliosides CCF52 and CCF4 however, showed significant suppression of IFN-γ expression indicating inhibition of T cell effector function at concentrations as low as 1μg/ml as shown in S1 Fig. Cell membrane blebbing and nuclear condensation were used as an index of apoptotic cell morphology [40]. As seen in Fig 1C, fluorescent microscopy revealed membrane blebbing and nuclear condensation characteristic of ongoing apoptosis (Fig 1C) in T cells treated with GBM derived gangliosides isolated from three different tumor lines, CCF52, CCF4 and U87 at 72hrs. Fig 1D shows photomicrograph of a single field representing induction of nuclear blebbing in T cells exposed to GBM gangliosides.


GBM Derived Gangliosides Induce T Cell Apoptosis through Activation of the Caspase Cascade Involving Both the Extrinsic and the Intrinsic Pathway.

Mahata B, Biswas S, Rayman P, Chahlavi A, Ko J, Bhattacharjee A, Li YT, Li Y, Das T, Sa G, Raychaudhuri B, Vogelbaum MA, Tannenbaum C, Finke JH, Biswas K - PLoS ONE (2015)

GBM derived gangliosides induce apoptosis of T cells.Peripheral blood T lymphocytes were isolated and purified from blood of healthy volunteers by negative selection. T lymphocytes were co-cultured with 15μg/ml of CCF52 derived gangliosides for 18–72 hrs, followed by annexinV-PE/7AAD staining for flow cytometric estimation of apoptosis, as shown in Fig 1A (*p<0.05 vs Media, ***p<0.001 vs Media). Purified T cells were incubated with different concentration (1–10μg/ml) of CCF52 gangliosides for 48hrs and 72hrs and apoptosis was measured by flow cytometric analysis using annexinV-PE/7AAD staining as indicated in Fig 1B (*p<0.05 vs Media, ***p<0.001 vs Media). Fig 1C shows graphical representation showing the percentage of nuclear blebbing event in T cells treated with gangliosides (15μg/ml) isolated from different glioblastoma cell lines for 72hrs (*p<0.05 vs Media, ***p<0.001 vs Media). Fig 1D shows representative photomicrograph demonstrating induction of apoptosis in T cells following 72hrs treatment with three different GBM derived gangliosides (15μg/ml) as evidenced by microscopic evaluation of nuclear blebbing by staining with DAPI. Data represents mean of at least 3 independent experiments unless mentioned otherwise.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4520498&req=5

pone.0134425.g001: GBM derived gangliosides induce apoptosis of T cells.Peripheral blood T lymphocytes were isolated and purified from blood of healthy volunteers by negative selection. T lymphocytes were co-cultured with 15μg/ml of CCF52 derived gangliosides for 18–72 hrs, followed by annexinV-PE/7AAD staining for flow cytometric estimation of apoptosis, as shown in Fig 1A (*p<0.05 vs Media, ***p<0.001 vs Media). Purified T cells were incubated with different concentration (1–10μg/ml) of CCF52 gangliosides for 48hrs and 72hrs and apoptosis was measured by flow cytometric analysis using annexinV-PE/7AAD staining as indicated in Fig 1B (*p<0.05 vs Media, ***p<0.001 vs Media). Fig 1C shows graphical representation showing the percentage of nuclear blebbing event in T cells treated with gangliosides (15μg/ml) isolated from different glioblastoma cell lines for 72hrs (*p<0.05 vs Media, ***p<0.001 vs Media). Fig 1D shows representative photomicrograph demonstrating induction of apoptosis in T cells following 72hrs treatment with three different GBM derived gangliosides (15μg/ml) as evidenced by microscopic evaluation of nuclear blebbing by staining with DAPI. Data represents mean of at least 3 independent experiments unless mentioned otherwise.
Mentions: T cells were treated with gangliosides (15μg/ml) isolated from the GBM cell line CCF52 at varying time intervals (Fig 1A) or with varying ganglioside concentration (Fig 1B), followed by staining with annexinV and 7-AAD prior to FACS analysis. CCF52 gangliosides induced time dependent T cell apoptosis, as evidenced by increased annexinV+/7-AAD+ staining at 18hrs (20%) and reaching peak at 72hrs (32%) (Fig 1A). However, significant T cell apoptosis was only observed at 10μg/ml or higher concentration as shown in Fig 1B. Although, CCF52 gangliosides did not show any significant induction in T cell apoptosis at lower concentrations, both GBM gangliosides CCF52 and CCF4 however, showed significant suppression of IFN-γ expression indicating inhibition of T cell effector function at concentrations as low as 1μg/ml as shown in S1 Fig. Cell membrane blebbing and nuclear condensation were used as an index of apoptotic cell morphology [40]. As seen in Fig 1C, fluorescent microscopy revealed membrane blebbing and nuclear condensation characteristic of ongoing apoptosis (Fig 1C) in T cells treated with GBM derived gangliosides isolated from three different tumor lines, CCF52, CCF4 and U87 at 72hrs. Fig 1D shows photomicrograph of a single field representing induction of nuclear blebbing in T cells exposed to GBM gangliosides.

Bottom Line: GBM-ganglioside induced activation of the effector caspase-3 along with both initiator caspases (-9 and -8) in T cells while both the caspase-8 and -9 inhibitors were equally effective in blocking apoptosis (60% protection) confirming the role of caspases in the apoptotic process.Ganglioside-induced T cell apoptosis did not involve production of TNF-α since anti-human TNFα antibody was unable to protect T cells from nuclear blebbing and subsequent cell death.Thus, GBM-ganglioside can mediate T cell apoptosis by interacting with the TNF receptor followed by activation of both the extrinsic and the intrinsic pathway of caspases.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Medicine, Bose Institute, Kolkata, India.

ABSTRACT
Previously we demonstrated that human glioblastoma cell lines induce apoptosis in peripheral blood T cells through partial involvement of secreted gangliosides. Here we show that GBM-derived gangliosides induce apoptosis through involvement of the TNF receptor and activation of the caspase cascade. Culturing T lymphocytes with GBM cell line derived gangliosides (10-20 μg/ml) demonstrated increased ROS production as early as 18 hrs as indicated by increased uptake of the dye H2DCFDA while western blotting demonstrated mitochondrial damage as evident by cleavage of Bid to t-Bid and by the release of cytochrome-c into the cytosol. Within 48-72 hrs apoptosis was evident by nuclear blebbing, trypan blue positivity and annexinV/7AAD staining. GBM-ganglioside induced activation of the effector caspase-3 along with both initiator caspases (-9 and -8) in T cells while both the caspase-8 and -9 inhibitors were equally effective in blocking apoptosis (60% protection) confirming the role of caspases in the apoptotic process. Ganglioside-induced T cell apoptosis did not involve production of TNF-α since anti-human TNFα antibody was unable to protect T cells from nuclear blebbing and subsequent cell death. However, confocal microscopy demonstrated co-localization of GM2 ganglioside with the TNF receptor and co-immunoprecipitation experiments showed recruitment of death domains FADD and TRADD with the TNF receptor post ganglioside treatment, suggesting direct interaction of gangliosides with the TNF receptor. Further confirmation of the interaction between GM2 and TNFR1 was obtained from confocal microscopy data with wild type and TNFR1 KO (TALEN mediated) Jurkat cells, which clearly demonstrated co-localization of GM2 and TNFR1 in the wild type cells but not in the TNFR1 KO clones. Thus, GBM-ganglioside can mediate T cell apoptosis by interacting with the TNF receptor followed by activation of both the extrinsic and the intrinsic pathway of caspases.

No MeSH data available.


Related in: MedlinePlus